Fluorescence-based detection of single nucleotide permutation in DNA via catalytically templated reaction

Article abstract of DOI:10.1039/b709611a

Templated reduction of low fluorescence azidocoumarin-PNA conjugate to high fluorescence aminocoumarin was achieved using a catalytic amount of DNA with single nucleotide resolution. The Royal Society of Chemistry.

Full text of DOI:10.1039/b709611a

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Fluorescence-based detection of single nucleotide permutation in DNA
via catalytically templated reaction{
Zbigniew L. Pianowski and Nicolas Winssinger*
Received (in Cambridge, UK) 26th June 2007, Accepted 26th July 2007
First published as an Advance Article on the web 9th August 2007
DOI: 10.1039/b709611a
the azide reduction rather than phosphine oxidation as we found it
difficult to avoid partial phosphine oxidation in biologically
relevant conditions.9,16 For the oligonucleotide recognition,
peptide nucleic acids (PNAs)17,18 were deemed the optimal choice
as they are more resistant to enzymatic or chemical degradation
than natural oligonucleotides. Furthermore, several modifications
have been reported to enhance their cellular permeability19,20 and
short PNAs do not activate an antisense response, suggesting that
this approach may be compatible with cellular sensing of
oligonucleotides.
Templated reduction of low fluorescence azidocoumarin–PNA
conjugate to high fluorescence aminocoumarin was achieved
using a catalytic amount of DNA with single nucleotide
resolution.
Oligonucleotide-templated reactions have attracted tremendous
interest for their implication in prebiotic chemistry and as a means
to program and orchestrate designed reactions.1 An important
application of such reactions is the sequence specific detection of
DNA and RNA which is of paramount importance in biomedical
research.2 For oligonucleotide detection, it is desirable to have a
reaction where the template can turnover so to provide an
amplification of the DNA or RNA signal with a single nucleotide
resolution. Several strategies have already been reported notably
based on autoligation,3–6 hydrolysis,7,8 the Staudinger reaction9
and chemical ligations;10–12 however, few methods were demon-
strated to be catalytic in the template. Recently Seitz and
Grossmann reported a system based on a transthioesterification
to transfer a quencher group from one sequence to another with
good turnover;13 however, the impact of mutations was only
reported for a pyrimidine to purine mutation.
The suitably derivatized 7-azidocoumarin 3 was prepared
according to modifications of known procedures21,22 as summar-
ized in Scheme 1. Thus, treatment of 1 with 1,3-acetonedicar-
boxylic acid in 70% sulfuric acid followed by hydrolysis of the
ethyl carbamate afforded the 7-aminocoumarin 2. Diazotization of
2 followed by sodium azide displacement afforded the required
azidocoumarin 3 (51% yield for four steps).
In order to minimize the distance between the phosphine and
azide upon hybridization, a PNA backbone modification was used
to append the masked fluorophore at the C-terminus of the PNA.
As shown in Scheme 2, monomer 4-S, which has an N-(2-
aminoethyl)serine backbone with a TBS-protected side chain, was
loaded on Rink resin, affording resin 5 which was engaged in
standard automated Fmoc synthesis thus leading to 6. The silyl
group was then removed using TBAF and the fluorophore was
introduced via acylation. Cleavage of the final compounds with
TFA : meta-cresol (4 : 1) afforded the labelled PNAs 7. The
phosphine was introduced at the N-terminus of the PNA
oligomers 8 by coupling with 2-(diphenylphosphino)benzoic acid
and the oligomers were released from the polymer using TFA :
meta-cresol (4 : 1) thus affording phosphino–PNAs 9. For all
oligomers, two lysine residues were incorporated into the sequence
to improve water solubility and disrupt potential aggregations.
We next investigated the change in fluorescence upon unmask-
ing of the azidocoumarin 3 and its PNA conjugate 7 with excess
phosphine (1 mM tris(carboxyethyl)phosphine). It is known that
the fluorescence quantum yield of coumarins is strongly influenced
by substituents in the 7-position as it alters the relative energies
between the emissive state and the non-emissive state.23 Electron
Herein, we report the application of a system based on a masked
7-azidocoumarin–PNA conjugate which is unmasked via a
Staudinger reaction by a triphenylphosphine–PNA conjugate
(Fig. 1). The Staudinger reaction was seen as ideal based on its
documented compatibility with cellular chemistry.14,15 However,
we found it important that the change in fluorescence be linked to
Fig. 1 The catalytic cycle of the templated Staudinger reaction yielding a
fluorescent product.
Laboratoire de Chimie Organique et Bioorganique, Institut de Science et
d’Inge´nierie Supramole´culaires, Universite´ Louis Pasteur – CNRS, 8
alle´e Gaspard Monge, 67083 Strasbourg, France.
E-mail: winssinger@isis.u-strasbg.fr; Fax: +33 3 90 24 51 12
{ Electronic supplementary information (ESI) available: Experimental
procedure and additional kinetic measurements of templated reactions.
See DOI: 10.1039/b709611a
Scheme 1 Synthesis of 7-azidocoumarin-4-acetic acid 3.
3820 | Chem. Commun., 2007, 3820–3822
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Table 1 DNA template sequences
Symbol DNA sequence (59–39)
T1
Perfect match
T2
GGG TAT CCG AA GCA CGT CGG Perfect match with
two nucleotide
separation
T2mG GGG TAT GCG AA GCA CGT CGG C A G mutation
T2mA GGG TAT ACG AA GCA CGT CGG C A A mutation
T2mT GGG TAT TCG AA GCA CGT CGG C A T mutation
l = 360 nm). For all experiments, a two to one ratio of phosphine
9 to azide 7 was used to compensate for partial phosphine
oxidation which was observed.
The templated process was first investigated in 1 mM PBS
buffer (pH 7.0) using hexameric and octameric PNA probes at
concentrations ranging from 100–500 nM of azidoPNA. In all
cases, the reaction was very fast (nearly 30–35% conversion within
15 min) while the control reaction without DNA or using a
random DNA sequence did not proceed at all. Furthermore, the
reaction kinetic was slower for a single mismatch and the
conversion exceeded template loading using catalytic amounts of
template.24
We have previously noted that the fidelity and affinity of PNA/
DNA hybridization can be enhanced using formamide solution in
the context of microarray hybridizations involving PNAs.26 The
effect of formamide is due to a change in the relative contribution
of hydrogen bonding and hydrophobic stacking of the nucleotides
in the helices.27 We thus turned our attention to the potential of
the templated reaction involving PNA in a formamide solution.
We found that the templated reaction was faster than in water
(over 50% conversion within 15 min with 1 equivalent of template)
and more importantly that the template turned over faster giving
over 15% conversion within 15 min using just 1% of template
(Fig. 2). Using an 8-mer PNA with one equivalent of template, the
reaction rate was not particularly sensitive to the distance between
the two reactive moieties,24 consistent with the fact that the rate of
dissociation of the PNA is slow relative to the rate of the
Staudinger reaction under these conditions.1,28 Next, we looked at
the impact of single nucleotide mutation on the rate of reaction
using catalytic amounts of template (5%). The reaction rates were
significantly reduced for all mutations, with the C to A mutation
having the lowest rate of reaction and the C to T mutation having
Scheme 2 Synthesis of coumarin–PNA conjugate 7 and phosphine–
PNA conjugate 9.
donor substituents at the 7-position and polar solvents lower the
energy level of the emissive state relative to the non-emissive state
thereby increasing the quantum yield of the fluorophore.
Accordingly, a 22-fold increase in fluorescence was observed upon
reduction of azidocoumarin 3 (for ex 360 nm, em 455 nm) while a
7-fold increase in fluorescence was observed for the reduction of
azidocoumarin–PNA conjugate 7.24 The lower increase in
fluorescence with the PNA conjugate was attributed to physico-
chemical interactions between the nucleobases and the coumarin.25
While this reaction was very fast with a phosphine at mM
concentration, the reaction rate was slow between 10 mM and 1 mM
concentration and negligible below 500 nM.
Next, we investigated the ability of DNA to template the
reaction. For this purpose, we used an 18-mer single strand DNA
sequence with overhangs on both sides (T1, Table 1) as well as
sequences containing two to six residues between the hybridization
sites or single mutations and a random template. The PNAs were
incubated with the templates and the fluorescence emission level
was monitored as a function of time (em l = 455 nm; ex
Fig. 2 Reactions with octameric PNA 7 (500 nM) and 9 in 1 : 1
formamide : 1 mM PBS (37 uC) using 0 to 100% of template.
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be useful in DNA templated synthesis. The masked fluorogenic
properties of 7-azidocoumarin which can be unveiled with a
Staudinger reaction was demonstrated for DNA-templated
reactions and should be of utility beyond the present work. The
remarkable selectivity for single base pair mismatch and speed of
analysis (10–15 min) should find applications in diagnostics.
This work was in part supported by a grant from Human
Frontier Science Program (HFSP 80/2003) and a fellowship
(MNRT) from the French ministry of research and education. The
authors thank L. Oswald for her technical assistance.
Notes and references
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Fig. 3 Reactions with hexameric PNA 7 (500 nM) and 9 in 1 : 1
formamide : 1 mM PBS (37 uC) using 0 to 100% of template (top) and
with templates containing a single nucleotide mismatch (bottom).
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the highest rate. These results are consistent with the fact that a
pyrimidine to purine mutation has a greater destabilization effect
on the duplex due to the steric clash of the two purines from the
mismatch strands. Importantly, the reaction was at least three
times as fast for the perfect match template than for the template
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using just 1% template (1 nM).24
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Using hexameric PNAs 7 and 9 rather than the aforementioned
octamers under the same conditions still afforded a very fast
templated reaction (over 50% conversion within 15 min); however,
the distance between the two PNA fragments became significant,
suggesting that the rate of the Staudinger reaction was no longer
much faster than the rate of dissociation.24 Under these conditions,
a distance of two to four residues between the reacting PNAs was
optimal. As with the 8-mer PNA, the reaction was found to
still proceed with 1% template (Fig. 3 top). Most impressively, a
10-fold difference in fluorescence was observed for the perfect
match template relative to single nucleotide mismatched templates
within 15 min using catalytic amounts of template (Fig. 3 bottom).
In conclusion, we have developed a system based on a templated
Staudinger reaction which can be used to detect single strand
DNA down to 1 nM with single nucleotide resolution and signal
amplification by virtue of the template turnover. The use of
formamide to modulate the affinity of the PNA–DNA interaction
was found to be important to achieve good selectivity and should
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P. Kuzmic, J. L. Harris and N. Winssinger, ChemBioChem, 2006, 7,
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27 A. Sen and P. E. Nielsen, Nucleic Acids Res., 2007, 35, 3367–3374.
28 Z. J. Gartner, M. W. Kanan and D. R. Liu, Angew. Chem., Int. Ed.,
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3822 | Chem. Commun., 2007, 3820–3822
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